This blog is meant to allow Fragment-based Drug Design Practitioners to get together and discuss NON-CONFIDENTIAL issues regarding fragments.

01 December 2014

Fragments finger a PHD finger

As Teddy recently observed, epigenetics is big, and
fragments have played an important role against several targets. One class of
proteins that has received less attention is the group of PHD fingers, which
recognize methylated lysine residues. The pygo-BCL9 complex contains a PHD
finger that binds to a specific methylated lysine residue on histones, and has
been implicated in cancer. Marc Fiedler, Mariann Bienz and colleagues at the
MRC Laboratory in the UK
describe their efforts against this target in a new paper in ACS Chem. Biol.

The researchers started with a virtual screen of 225,000
commercially available compounds. They purchased 313 of the top hits and tested
them for binding with protein-detected NMR (1H-15N-HSQC).
This produced only three very weak hits – a hit rate of 0.001%. Three
additional virtual screens produced a couple dozen more, but all of these were
weak; the best had an affinity around 3.5 mM and a ligand efficiency around
0.12 kcal/mol/atom. Co-crystallography proved unsuccessful, probably in part
due to the low solubility of the compounds.

Enter fragments. The researchers screened the Maybridge
“rule of three” 1000-compound library in pools of 5 compounds, each at 1 mM, under
the same protein-detected NMR conditions they used previously. Numerous pools
appeared to show binding but deconvolution proved unsuccessful for all but two.
Strikingly, the two hits – both benzothiazoles – are almost identical,
differing only in a single atom substituent (fluorine vs chlorine).

Although the best fragment hit was also weak (Kd
= 3.1 mM), it had a much higher ligand efficiency (0.31 kcal/mol/atom). More
importantly, it was sufficiently soluble (20 mM!) that it could be
cocrystallized with the protein, resulting in a high resolution structure. This
revealed that the fragment binds in a narrow cleft – a conclusion independently
reached by examining the NMR chemical shift perturbations (CSPs) of protein
amino acid residues in the presence of compound.

Testing various analogs did not identify anything
significantly more potent, but changing the benzothiazole core to a
benzimidazole changed the pattern of CSPs. Additional NMR studies and modeling
suggested that these molecules bind not in the narrow cleft but rather in the
pocket where methylated lysine binds, and competition studies with a short
peptide supported this hypothesis.

This is a nice example of applying fragments against an
important emerging target class. It is also a beautiful illustration of
molecular complexity in action: as the authors note, the hit rate from fragment
screening was around 200-fold higher than the virtual screen, and provided better hits to boot. As with most
fragment screens there is still a long way to go to get to a potent compound, but it looks like this group is on the right path.